Abstract

The opportunistic fungal pathogen Candida albicans thrives within diverse niches in the mammalian host. Among the adaptations that underlie this fitness is an ability to utilize a wide array of nutrients, especially sources of carbon that are disfavored by many other fungi; this contributes to its ability to survive interactions with the phagocytes that serve as key barriers against disseminated infections. We have reported that C. albicans generates ammonia as a byproduct of amino acid catabolism to neutralize the acidic phagolysosome and promote hyphal morphogenesis in a manner dependent on the Stp2 transcription factor. Here, we report that this species rapidly neutralizes acidic environments when utilizing carboxylic acids like pyruvate, α-ketoglutarate (αKG), or lactate as the primary carbon source. Unlike in cells growing in amino acid-rich medium, this does not result in ammonia release, does not induce hyphal differentiation, and is genetically distinct. While transcript profiling revealed significant similarities in gene expression in cells grown on either carboxylic or amino acids, genetic screens for mutants that fail to neutralize αKG medium identified a nonoverlapping set of genes, including CWT1, encoding a transcription factor responsive to cell wall and nitrosative stresses. Strains lacking CWT1 exhibit retarded αKG-mediated neutralization in vitro, exist in a more acidic phagolysosome, and are more susceptible to macrophage killing, while double cwt1Δ stp2Δ mutants are more impaired than either single mutant. Together, our observations indicate that C. albicans has evolved multiple ways to modulate the pH of host-relevant environments to promote its fitness as a pathogen.

IMPORTANCE:

The fungal pathogen Candida albicans is a ubiquitous and usually benign constituent of the human microbial ecosystem. In individuals with weakened immune systems, this organism can cause potentially life-threatening infections and is one of the most common causes of hospital-acquired infections. Understanding the interactions between C. albicans and immune phagocytic cells, such as macrophages and neutrophils, will define the mechanisms of pathogenesis in this species. One such adaptation is an ability to make use of nonstandard nutrients that we predict are plentiful in certain niches within the host, including within these phagocytic cells. We show here that the metabolism of certain organic acids enables C. albicans to neutralize acidic environments, such as those within macrophages. This phenomenon is distinct in several significant ways from previous reports of similar processes, indicating that C. albicans has evolved multiple mechanisms to combat the harmful acidity of phagocytic cells.

Medium neutralization induced by growth on carboxylic acids does not generate ammonia. The wild-type SC5314 strain was spotted onto solid YNB medium with the indicated compound as the sole carbon source and allowed to develop into a colony at 37°C. Directly apposed to the colony, a small reservoir was affixed to the lid of the petri dish and filled with 10% citric acid. At the indicated times, a sample of the liquid in the acid trap was removed and assayed for nitrogen content using Greiss reagent; the results are expressed as parts per million (ppm). CAA, Casamino Acids; Glut, glutamate; Ser; serine; Pyr, pyruvate; αKG, α-ketoglutarate; Ace, acetate; Lac, lactate. Error bars show standard deviations.

Extracellular neutralization on carboxylic acids does not induce hyphal germination. Cells of the wild type SC5314 strain were grown overnight in YPD and then washed, diluted into YNB with the indicated compound present as the sole carbon source (2% wt/vol), and grown at 37°C for the indicated times before being fixed and imaged. The number in white in each image is the pH of that culture at that time point. CAA, Casamino Acids; αKG, α-ketoglutarate; Pyr, pyruvate; Ace; acetate; Lac, lactate. The scale bar in the lowest image on the right is 10 µm.

Transcriptional changes in response to amino acids or α-ketoglutarate are substantially similar. Transcriptional profiles were assessed using RNA sequencing of cells grown in minimal YNB medium containing glucose, Casamino Acids (CAA), glutamate (Glut), or α-ketoglutarate (αKG) for 5 to 7 h at 37°C. Ratios of transcript abundance based on the reads per kilobase per million (FPKM) metric under each condition relative to the expression in the glucose control were used to determine differentially regulated genes. (A) K-means clustering of differentially regulated genes demonstrates the similar changes under the three conditions. (B) Venn diagram of the overlap between the three conditions in genes upregulated by at least threefold. (C) Downregulated genes.

cwt1Δ mutants fail to germinate or fully neutralize the phagosome. (A) FITC-labeled strains of the indicated genotypes (see the legend to ) were incubated with J774A.1 macrophages for 1 h before fixing and staining with Calcofluor white to discriminate between extracellular and intracellular C. albicans cells. The morphology of phagocytosed cells only was scored and plotted. Error bars show standard deviations. (B) The indicated strains were incubated with LysoTracker red (LR)-loaded J774A.1 cells for 1 h before fixation. LR intensity in the 10 pixels (1 µm) immediately adjacent to the fungal cell along a line perpendicular to the cell was averaged for at least 50 cells per strain and plotted in the box-and-whisker plot, where the whiskers show the 5th to 95th percentiles. (C) Representative images are shown in which the fungal cells are labeled with FITC (green), while the macrophages are loaded with LR (red). Calcofluor white (blue) is used to differentiate external C. albicans cells (which stain blue) from phagocytosed cells (which do not stain). Asterisks represent a P value of <0.05 for the indicated comparison.

Cwt1 contributes to fungal survival in macrophages. (A) The survival of the indicated strains (see the legend to ) was assessed using an endpoint dilution assay after coculture with J774A.1 cells for 24 h and expressed relative to the survival of cells cultured in the absence of macrophages. (B) The release of lactate dehydrogenase (LDH) from macrophages was assayed as a measure of fungus-induced membrane damage and expressed relative to the release of LDH from chemically lysed macrophages. *, P < 0.05 relative to the wild-type control strain. Error bars show standard deviations.